Activation of the Tumor Necrosis Factor Receptor-1 (TNFR-1) by the ligand TNF initiates two major intracellular signaling pathways that lead to the activation of the transcription factor NFκB (Tartaglia & Goeddel, 1992) and the induction of cell death (Grell et al., 1993; Tartaglia et al., 1993). Interaction of a TNF trimer with a TNFR-1 trimer induces intracellular self-association of the TNFR-1 death domain (DD) (Smith et al., 1994) (Chan et al., 2000) allowing for the recruitment of an adaptor protein named TNFR-Associated Death Domain protein (TRADD) through a death domain/death domain interaction. TRADD (Hsu et al., 1995) recruits the signaling molecule TNFR-Associated Factor-2 (TRAF-2) (Hsu et al., 1996b) through interactions with the N-terminal domain. On the other hand, Fas Associated Death Domain protein (FADD) (Hsu et al., 1996b) and the Receptor Interacting Protein (RIP) (Hsu et al., 1996a) are recruited to the TNFR-1 signaling complex through death domain interactions with TRADD.
RIP is required for activation of the transcription factor NFκB by TNF (Kelliher et al., 1998) and is a structurally unique protein containing both a kinase domain and a death domain (Stanger et al., 1995). Overall, the protein contains 671 residues and three domains consisting of a 300 residue N-terminal serine/threonine kinase domain, a 272 residue intermediate region, and a 99 residue death domain. Deletion mutagenesis studies have shown that the N-terminal region of RIP is involved in binding to TRAF-2 and that the intermediate region of RIP is the critical domain for activation of the transcription factor NFκB (Hsu et al., 1996a). The deletion mutagenesis studies also showed that RIP DD binds to TRADD DD allowing for the recruitment of RIP to the TNFR-1 signaling complex. Importantly, it has been shown that RIP DD can block TNF-mediated NFκB and JNK activation, probably by competing with endogenous RIP for interaction with the TNFR-1/TRADD complex (Hsu et al., 1996a). RIP was also found to interact with RAIDD, which is another adaptor molecule in the ICH-1 pathway, through a death domain/death domain interaction (Duan & Dixit, 1997).
Structural studies, especially by NMR, on death domain proteins at physiological pH have been complicated by the tendency of the proteins to self-associate and form large molecular weight aggregates. This is also the case for the Death Effector Domains (DED) and the Caspase Recruiting Domains (CARD) which are structurally related to the death domains. Therefore, structural studies of the death domain superfamily of proteins requires relatively low (≦4) or high (≧8) pH to minimize the natural tendency of self aggregation. This was the case for FAS DD (Huang et al., 1996), FADD DED (Eberstadt et al., 1998), FADD DD (Jeong et al., 1999), RAIDD CARD (Chou et al., 1998), TNFR-1 DD (Telliez et al., 2000), and in the current study of the RIP DD. In addition, single point mutants which alter protein solubility were necessary for the structural studies of the FADD DED (Chou et al., 1998) and TNFR-1 DD (Telliez et al., 2000). To date, the structures of several death domains have been solved: FAS DD (Huang et al., 1996), p75 neurotrophin DD (Liepinsh et al., 1997), FADD DD (Jeong et al., 1999), a complex structure of the Tube DD with the Pelle DD (xiao et al., 1999) and TNFR-1 DD (Sukits, et al., 2001). In addition, one death effector domain structure (FADD DED) (Eberstadt et al., 1998), and three CARD domain structures: RAIDD CARD (Chou et al., 1998), APAF-1 (Zhou et al., 1999) (Qin et al., 1999), and the complex between APAF-1 and procaspase-9 (Zhou et al., 1999) have been solved. All the structures have the same general fold consisting of a core of six α-helices arranged in an anti-parallel fashion, where the lengths and orientations of the helices are slightly different in the various structures.